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Probe, primer and detection kit for detecting C-KIT gene mutation

A detection kit, the technology of the kit, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of long detection time, false negative, low sensitivity, etc., and achieve fast detection speed. , the effect of strong specificity and high sensitivity

Active Publication Date: 2013-05-22
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity of direct sequencing is low, and the detection sensitivity is only 20-30%. Low-sensitivity detection will lead to missed detection and false negatives
In addition, the direct sequencing method has complex experimental operations, low detection efficiency, easy sample contamination, and long detection time, which cannot meet the actual needs of clinical detection. There is an urgent need to develop a high-sensitivity and rapid C-KIT gene mutation detection technology in order to realize Use high-sensitivity detection method to detect C-KIT gene mutation, so as to provide scientific reference for individualized treatment of clinical tumors

Method used

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  • Probe, primer and detection kit for detecting C-KIT gene mutation
  • Probe, primer and detection kit for detecting C-KIT gene mutation
  • Probe, primer and detection kit for detecting C-KIT gene mutation

Examples

Experimental program
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Embodiment 1

[0037] In this embodiment, the plasmid constructed by genetic engineering is used as the positive plasmid, and the method for detecting the D842V mutation of the C-KIT gene by real-time fluorescent PCR of the present invention includes the following steps:

[0038] (1) Detection sample DNA extraction

[0039] Add 1 mL of xylene to the sample for dewaxing, centrifuge to collect the precipitate, add 1 mL of absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, digest and lyse at 56°C for 1 hour, incubate at 90°C for 1 hour, add 200mL Mix Buffer AL, then add 200 μL absolute ethanol and mix well, carefully transfer the supernatant to a QIA 2mL spin column, centrifuge at 6000×g (8000rpm) for 1min, add 500μL BufferAW1, centrifuge at 6000×g (8000rpm) for 1min, carefully open the lid and add Centrifuge 500μL Buffer AW2 at 6000×g (8000rpm) for 1min, centrifuge the empty tube at 20000×g (14000rpm) for 3min, add 100μL Buffer ATE to the cen...

Embodiment 2

[0051] Using the present invention to detect clinical samples, a total of 90 clinical gastrointestinal stromal tumor samples from our company were taken, including 40 males and 50 females, aged 30-72 years old, with an average age of 50 years old. The steps of detecting C-KIT gene mutations in 90 cases of clinical samples by using the real-time fluorescent PCR system of the present invention are as follows.

[0052] (1) Detection sample DNA extraction

[0053] Test samples included fresh pathological tissue, paraffin-embedded tissue, whole blood, plasma, and pleural effusion. For fresh pathological tissue and paraffin tissue samples, cut the sample into 5-10 μm, add 1 mL of xylene to dewax, collect the precipitate by centrifugation, add 1 mL of absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and BufferATL, 56 Digest and lyse at ℃ for 1 hour, incubate at 90°C for 1 hour, add 200 mL of Buffer AL to mix, then add 200 μL of absolute ethanol ...

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Abstract

The invention discloses a probe, a primer and a detection kit for detecting C-KIT gene mutation, which comprise the following sequence: SEQ ID NO1-SEQ ID NO3. By utilizing a specific primer and a probe technology, a real-time fluorescent PCR (polymerase chain reaction) system for detecting C-KIT gene mutation is established. The method has the advantages that (1) the sensitivity is high, and 5-10 copied mutation DNA (deoxyribonucleic acid) can be detected; (2) the specificity is strong, 10ng of wild type genome DNA cannot generate non-specific signals; and (3) the detection speed is high, the detection can be finished only in 90 minutes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a C-KIT gene mutation detection probe, primer and kit. Background technique [0002] C-KIT is a proto-oncogene located on chromosome 4q11-12 with a total length of about 80kb. The C-KIT protein encoded by C-KIT belongs to the type III receptor tyrosine kinase family member, and its molecular weight is about 145KD transmembrane protein, including the intracellular tyrosine kinase region, transmembrane region and ligand binding site Point extracellular region. When the ligand binds to it, it can activate its own tyrosine protein kinase activity, and activate the downstream signal transduction pathway through a series of reactions, thereby regulating the growth and proliferation of cells. [0003] Gleevec (Gleevec) is a targeted drug developed for C-KIT, which blocks the phosphate group from ATP to the substrate tyrosine by binding to the ATP site in the cytoplasmic tyrosine kinase fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 阮力林清华罗捷敏郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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